Treatment of inflammatory conditions of the intestine

ABSTRACT

A method for the treatment and/or prophylaxis of an inflammatory condition of the intestine of a patient, comprises parenteral administration to the patient of an effective amount of high density lipoprotein (HDL)

FIELD OF THE INVENTION

This invention relates generally to a method for the treatment and/orprophylaxis of inflammatory conditions of the intestine, including butnot limited to inflammation and inflammatory damage associated withischaemia/reperfusion injury of the intestine, inflammatory boweldisease and colitis. More particularly, the present invention relates tothe use of high density lipoproteins (HDLs) in the treatment and/orprophylaxis of these inflammatory conditions of the intestine.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to in thisspecification are referenced at the end of the description. Thereference to any prior art document in the specification is not, andshould not be taken as, an acknowledgment or any form of suggestion thatthe document forms part of the common general knowledge in Australia.

High-density lipoproteins (HDLs) represent a broad group of mostlyspheroidal plasma lipoproteins, which exhibit considerable diversity intheir size, apolipoprotein (apo) and lipid composition. HDL particlesfall into the density range of 1.063-1.21 g/ml (1) and as they aresmaller than other lipoproteins, HDLs can penetrate between endothelialcells more readily allowing relatively high concentrations to accumulatein tissue fluids (2). The major apolipoprotein of almost all plasma HDLsis apo A-1, which in association with phospholipids and cholesterol,encloses a core of cholesteryl esters (1). Nascent (i.e. newlysynthesised) HDLs secreted by the liver and intestine contain nocholesteryl esters and are discoidal in shape (1). The negativeassociation of plasma HDL concentration with coronary artery disease hasbeen well documented in epidemiological studies (3). Althoughexperiments in animals have demonstrated an anti-atherogenic activity ofHDLs (4), it is not yet known whether this protective effect is relatedto the role of the lipoprotein in reverse cholesterol transport or to adifferent mechanism. The mechanism/mechanisms via which HDLs providethese cardioprotective actions are not clearly understood, but mayinclude a role for HDLs in reverse transport of cholesterol fromperipheral tissues to the liver, inhibition of the oxidation oflow-density lipoproteins, or modulation of vasodilatation and plateletactivation mediated by changes in the production of prostacyclin (5).HDLs can also activate endothelial nitric oxide synthase subsequent toits interaction with scavenger receptor-B1 (SR-B1). Although HDLs areinvolved in the removal of cholesterol from extra-hepatic tissues, thissubset of lipoproteins has recently been reported to possess functionsunrelated to their role in plasma cholesterol transport. Almost 10 yearsago, it was reported that in transgenic mice in which plasma levels ofHDL were two-fold higher, the increase in plasma levels of TNF-α as wellas mortality caused by bacterial lipopolysaccharide (LPS) weresignificantly reduced (6). Subsequently, it has been demonstrated thatadministration of native HDL or reconstituted HDL (=recHDL)significantly reduces organ injury and levels of mortality in animalmodels of endotoxic (LPS-mediated) and haemorrhagic shock (7). Thebeneficial actions observed in these models are—at least inpart—mediated by the ability of HDLs to bind and inactivate LPS (6,8),directly inhibit expression of adhesion molecules on endothelial cellsand via modulation of the expression of proinflammatory cytokines (6,9).In human volunteers, systemic administration of HDLs also downregulatesthe LPS ligand CD14 on monocytes and attenuates the release of TNF-α,IL-6 and IL-8 caused by small doses of intravenously administered LPS(10). HDL has also been shown to directly inhibit the TNF-α-inducedexpression of P-selectin on human endothelial cells (6). In addition, ithas been reported that HDLs reduces the renal injury, dysfunction andinflammation caused by bilateral renal artery occlusion and reperfusionin the rat (11).

A growing body of data indicates that oxygen-derived free radicals suchas superoxide (O₂—), nitric oxide (NO) and hydroxyl radicals (OH—) havea role in mediating the intestinal damage in ischaemia/reperfusion [I/R](12,13) as well as in inflammatory bowel disease (IBD) (14). Theintestine is well endowed with enzymes capable of producing such freeradicals (15). Moreover, when inflammation is present the manyphagocytic cells that are attracted and activated can produce largeamounts of free radicals. Several studies suggest that peripheral bloodmonocytes (16), and isolated intestinal macrophages (17), from patientswith IBD produce increased amounts of free radicals. Also high numbersof peripheral polymorphonuclear leukocytes (PMNs), which are capable ofproducing large amounts of oxygen-derived free radicals (18), migrateinto the intestinal wall of such patients (19). Grisham and Granger (20)hypothesised that—like in I/R injury—in ulcerative colitis transientischaemic and subsequent reperfusion produce high levels of freeradicals. This process initiates a cascade of events leading to therecruitment and activation of PMNs. In the last few years, variousstudies have gained substantial insight into the importance of specificadhesion molecules and mediators in processes, which finally result inthe recruitment of PMNs at a specific site of inflammation. ActivatedPMNs, therefore, play a crucial role in the destruction of foreignantigens and the breakdown and remodelling of injured tissue.PMN-endothelial interactions involve a complex interplay among adhesionglycoproteins (i.e. integrins, members of the immunoglobulin superfamilyand selectins). The firm adhesion of PMNs to the endothelium, however,is a complex phenomenon, which also involves other endothelium-basedadhesion molecules. In fact, endothelial adhesion molecules areconsidered to play a pivotal role in the localisation and development ofan inflammatory reaction (21). Intercellular adhesion molecule-1(ICAM-1) is an adhesion molecule normally expressed at a low basallevel, but its expression can be enhanced by various inflammatorymediators such as TNF-α and IL-1β (22).

Models of splanchnic artery occlusion shock (SAO) and2,4,6-dinitrobenzene-sulfonic acid (DNBS)-induced colitis have beenwidely employed to investigate the pathophysiology of intestinal damageassociated with I/R and with IBD. In work leading to the presentinvention, the inventors have investigated whether recHDL reduces theintestinal injury and inflammation caused by SAO shock and the chronicinflammatory response (colitis) caused by injection of DNBS in the rat.In order to highlight the possible mechanisms through which HDLs conferprotection, the following endpoints of the inflammatory response havebeen determined: (1) PMN infiltration (determined as myeloperoxidase(MPO] activity, (2) pro-inflammatory cytokine production, (3) expressionof adhesion molecules (i.e. ICAM-1), (4) lipid peroxidation (evaluatedas malondialdehyde (MDA] levels) (5) peroxynitrite formation, (6)activation of the nuclear enzyme poly (ADP-ribose) (PAR) polymerase(PARP) and (7) morphological changes in the intestine.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and or variations suchas “comprises” or “comprising”, will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

In one aspect, the present invention provides a method for the treatmentand/or prophylaxis of an inflammatory condition of the intestine of apatient, which comprises parenteral administration to the patient of aneffective amount of high density lipoprotein (HDL).

In another aspect, the present invention provides the use of highdensity lipoprotein (HDL) in the manufacture of a medicament forparenteral administration to a patient for the treatment and/orprophylaxis of an inflammatory condition of the intestine of thepatient.

In yet another aspect, the invention provides an agent for parenteraladministration in the treatment and/or prophylaxis of an inflammatorycondition of the intestine of a patient, which comprises high densitylipoprotein (HDL).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of HDL treatment on mean blood pressure andsurvival. Effect of HDL treatment on MAP (A) and mortality (B). Nosignificant alteration of MAP was observed in sham-operated rats. Fallin MAP and the mortality in SAO rats was significantly reduced by HDLtreatment (80 mg/kg). Values are means±S.E.M. of 10 rats for each group.*P<0.01 versus sham, °P<0.01 versus I/R.

FIG. 2 shows MDA and MPO tissue levels. Reperfusion of the ischaemicsplanchnic circulation leads to profound increase in MDA levels (A) andin MPO (B) in ileum tissues which is inhibited by HDL treatment (80mg/kg). Values are means±S.E.M. of 10 rats for each group. *P<0.01versus sham, °P<0.01 versus I/R.

FIG. 3 shows plasma levels of TNF-α and IL-1β. Reperfusion of theischaemic splanchnic circulation leads to profound increase in plasmaTNFα and IL-1β production and this is inhibited by HDL (80 mg/kg).Values are means±S.E.M. of 10 rats for each group. *P<0.01 versus sham,°P<0.01 versus I/R.

FIG. 4 shows immunohistochemical staining of ICAM-1. I/R induced anincrease of the positive staining for ICAM-1 along the endothelium wall(A). In HDL-treated rats (B) subjected to SAO-shock, there was noincrease of immunostaining for ICAM-1, which was present only along theendothelium wall. Original magnification: ×500. Figure is representativeof at least 3 experiments performed on different experimental days.

FIG. 5 shows typical Densitometry evaluation. Densitometry analysis ofImmunocytochemistry photographs (n=5) for ICAM-1, nitrotyrosine and PARfrom ileum (A) and from colon (B).was assessed. The assay was carriedout by using Optilab Graftek software on a Macintosh personal computer(CPU G3-266). Data are expressed as % of total tissue area. *P<0.01versus Sham. °P<0.01 versus IR or versus DNBS.

FIG. 6 shows immunohistochemical staining of nitrotyrosine and PAR.After reperfusion nitrotyrosine (A) and PAR (C) staining was localisedin the injured area from a SAO-shocked rat. There was no detectableimmunostaining for nitrotyrosine (B) and PAR (D) in the ileum fromHDL-treated rats. Original magnification: ×500. Figure is representativeof at least 3 experiments performed on different experimental days.

FIG. 7 shows the effect of HDL treatment on the tissue damage. Distalileum section from SAO shocked-rats showed inflammatory cellinfiltration extending through the wall and concentrated below theepithelial layer and demonstrating oedema of the distal portion of thevilli (A). Distal ileum from HDL-treated rats (B) shows reducedSAO-induced organ injury. Original magnification: ×125. Figure isrepresentative of at least 3 experiments performed on differentexperimental days.

FIG. 8 shows the effect of HDL treatment on the damage score and oncolon injury. Colonic damage (A) was scored on a 0 (normal) to 10(severe) scale by two independent observers. Histological examination ofdescending colon from DNBS-treated rats (B) reveals a completealteration of the epithelial layer, muscularis mucosa and submucosal aswell as a diffuse inflammatory cells infiltration in perilesional area.Treatment with HDL (C) significantly reduced the damage score (A) andcorrected the disturbances in morphology and reduced the inflammatorycells infiltration associated with DNBS administration. Originalmagnification: ×100. Figure is representative of at least 3 experimentsperformed on different experimental days. Values are means±S.E.M. of 10rats for each group. *P<0.01 vs. sham; °P<0.01 vs. DNBS.

FIG. 9 shows organ weight. A significant increase was consistently seenat 4 days after DNBS injection in colon (A) and spleen (B). The weightof the organs was significantly reduced in the rats which had beentreated with HDL. Values are means±s.e. means of 10 rats for each group.*p<0.01 vs. sham; °p<0.01 vs. DNBS.

FIG. 10 shows the effect of HDL treatment on body weight changes andTNF-α and IL-1β levels at 4 days after DNBS intracolonic administration.A significant loss of body weight (A) and an increase of TNF-α and IL-1β(B) were observed in the DNBS-treated rats. HDL treatment significantlyprevented the loss of body weight and reduced the increase of cytokinelevels in the colon. Values are means±S.E.M. of 10 rats for each group.*P<0.01 vs. sham; °P<0.01 vs. DNBS.

FIG. 11 shows immunohistochemical localisation for nitrotyrosine and forpoly (ADP-ribose) in the colon. Immunohistochemical for nitrotyrosine(A) and for poly (ADP-ribose) (C) show positive staining primarilylocalised in the infiltrated inflammatory cells and in disruptedepithelial cells from a DNBS treated rats. The intensity of the positivestaining for nitrotyrosine (B) and for poly (ADP-ribose) (D) wassignificantly reduced in the colon from HDL-treated rats. Originalmagnification: ×250. Figure is representative of at least 3 experimentsperformed on different experimental days.

FIG. 12 shows the effect of HDL on neutrophil infiltration and lipidperoxidation. Malondialdehyde (MDA) (A) and myeloperoxidase (MPO)activity (B) in the colon from DNBS-treated rats. MPO activity and MDAlevels were significantly increased in DNBS-treated rats in comparisonto sham. HDL-treated rats show a significant reduction of MPO activityand MDA levels. Values are means±s.e. means of 10 rats for each group.*p<0.01 vs. sham; °p<0.01 vs. DNBS.

FIG. 13 shows immunohistochemical localisation of ICAM-1 in the colon.Colon section obtained from DNBS-treated rats showed intense positivestaining for ICAM-1 (A) on the vessels as well as in inflammatory cellsconcentrated below the epithelial layer. The degree of positive stainingfor ICAM-1 (B) was markedly reduced in tissue section obtained fromHDL-treated rats. Original magnification: ×250. Figure is representativeof at least 3 experiments performed on different experimental days.

DETAILED DESCRIPTION OF THE INVENTION

High-density lipoproteins (HDLs) have been shown to reduce the organinjury and mortality in animal models of shock by reducing theexpression of adhesion molecules and pro-inflammatory enzymes. However,there is limited evidence that HDL treatment reduces inflammation. Asinflammation plays an important role in the development of colitis aswell as ischaemia/reperfusion (I/R) injury of the intestine, theinventors have investigated the effects of HDL in animal modelsassociated with gut injury and inflammation [splanchnic artery occlusion(SAO) shock and dinitrobenzene sulfonic acid (DNBS) induced colitis],and shown that the administration of reconstituted HDL (recHDL) (80mg/kg i.v. bolus 30 min prior ischaemia in the SAO-shock model or 40mg/kg i.v. every 24 h in the colitis model) exerts potentanti-inflammatory effects (e.g. reduced inflammatory cell infiltrationand histological injury, and delayed the development of the clinicalsigns) in vivo. Furthermore, recHDL reduced (i) the staining fornitrotyrosine and poly (ADP-ribose) (immunohistochemistry) and; (ii) theexpression of intercellular adhesion molecule-1 in the ileum ofSAO-shocked rats and in the colon from DNBS-treated rats. Thus, recHDLreduces the inflammation caused by intestinal I/R and colitis.

In one aspect, the present invention provides a method for the treatmentand/or prophylaxis of an inflammatory condition of the intestine of apatient, which comprises parenteral administration to the patient of aneffective amount of high density lipoprotein (HDL).

Reference herein to “treatment” or “prophylaxis” is to be considered inits broadest context. The term “treatment” does not necessarily implythat a subject is treated until total recovery. Similarly, “prophylaxis”does not necessarily mean that the subject will not eventually contracta disease condition. Accordingly, treatment and prophylaxis includeamelioration of the symptoms of a particular condition or preventing orotherwise reducing the risk of developing a particular condition. Theterm “prophylaxis” may be considered as reducing the severity or onsetof a particular condition. “Treatment” may also reduce the severity ofan existing condition.

Inflammatory conditions of the intestine to which the present inventionrelates include, but are not limited to, inflammation and inflammatorydamage associated with ischaemia/reperfusion (I/R) injury of theintestine, inflammatory bowel disease (including Crohn's disease andulcerative colitis), acute infective colitis, and pseudomembranouscolitis (an antibiotic-induced condition caused by Clostridiumovergrowth). Such diseases are described in relevant standard textbookswhich include Harrison's Principles of Internal Medicine, OxfordTextbook of Medicine, and Principles and Practice of Gastroenterologyand Hepatology.

In accordance with the present invention, HDL is administered to apatient. The term “HDL” as used herein relates to all forms of highdensity lipoproteins and includes nascent HDL or reconstituted HDL(RHDL) or any mixture thereof, as well as recombinant HDL or an analoguethereof with functional relationship to nascent or reconstituted HDL.Such analogues include functional peptides derived from theapolipoprotein (Apo) structure such as those described in InternationalPatent Publications Nos. WO 99/16459 and WO 99/16408, the contents ofwhich are incorporated herein by reference.

The high density lipoproteins comprise a protein component, and lipid.The proteins are preferably apolipoproteins, e.g. human apolipoproteinssuch as apolipoprotein A-I (apoA-II) or apolipoprotein A-II (apoA-II) orrecombinant apolipoproteins, or functionally homologous peptides withsimilar properties. Suitable lipids are phospholipids, preferablyphosphatidyl choline, optionally mixed with other lipids (cholesterol,cholesterol esters, triglycerides, or other lipids). The lipids may besynthetic lipids, naturally occurring lipids or combinations thereof.

Production of reconstituted HDL is described, by way of example, in U.S.Pat. No. 5,652,339 and by Matz and Jonas (24) and Lerch et al. (25).Production of recombinant HDL is described, by way of example, inEuropean Patent No. EP 469017 (in yeast), U.S. Pat. No. 6,559,284 (in E.coli), and International Patent Publications Nos. WO 87/02062 (in E.coli, yeast and Cho cells) and WO 88/03166 (in E. coli). The contents ofeach of these documents are incorporated herein by reference.Preferably, the HDL is reconstituted HDL.

The HDL is administered in an effective amount. An “effective amount”means an amount necessary at least partly to attain the desiredresponse, or to delay the onset or inhibit progression or haltaltogether, the onset or progression of the particular condition beingtreated. The amount varies depending upon the health and physicalcondition of the individual to be treated, the racial background of theindividual to be treated, the degree of protection desired, theformulation of the composition, the assessment of the medical situation,and other relevant factors. It is expected that the amount will fall ina relatively broad range that can be determined through routine trials.

Preferred HDL dosage ranges are from 0.1-200 mg, more preferably 10-80mg, HDL (weight based on apolipoprotein) per kg body weight pertreatment. For example, the dosage of HDL which is administered may beabout 0.2-100 mg HDL per kg body weight (weight based on apolipoprotein)given as an intravenous injection and/or as an infusion for a clinicallynecessary period of time, e.g. for a period ranging from a few minutesto several hours, e.g. up to 24 hours. If necessary, the HDLadministration may be repeated one or several times. The actual amountadministered will be determined both by the nature of the disease whichis being treated and by the rate at which the HDL is being administered.

Preferably, the patient is a human, however the present inventionextends to treatment and/or prophylaxis of other mammalian patientsincluding primates, livestock animals (e.g. sheep, pigs, cattle, horses,donkeys), laboratory test animals (e.g. mice, rabbits, rats, guineapigs), companion animals (e.g. dogs, cats) and captive wild animals.

In accordance with the present invention, the HDL is administered to apatient by a parenteral route of administration. Parenteraladministration includes any route of administration that is not throughthe alimentary canal (that is, not enteral), including administration byinjection, infusion and the like. Administration by injection includes,by way of example, into a vein (intravenous), an artery (intraarterial),a muscle (intramuscular) and under the skin (subcutaneous). The HDL mayalso be administered in a depot or slow release formulation, forexample, subcutaneously, intradermally or intramuscularly, in a dosagewhich is sufficient to obtain the desired pharmacological effect.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the active component which ispreferably isotonic with the blood of the recipient. This aqueouspreparation may be formulated according to known methods using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in a polyethylene glycol and lactic acid.Among the acceptable vehicles and solvents that may be employed arewater, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conveniently employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono- or di-glycerides. In addition, fatty acidssuch as oleic acid find use in the preparation of injectables.

The formulation of such therapeutic compositions is well known topersons skilled in this field. Suitable pharmaceutically acceptablecarriers and/or diluents include any and all conventional solvents,dispersion media, fillers, solid carriers, aqueous solutions, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like. The use of such media and agents forpharmaceutically active substances is well known in the art, and it isdescribed, by way of example, in Remington's Pharmaceutical Sciences,18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofaras any conventional media or agent is incompatible with the activeingredient, use thereof in the pharmaceutical compositions of thepresent invention is contemplated. Supplementary active ingredients canalso be incorporated into the compositions.

Other delivery systems can include sustained release delivery systems.Preferred sustained release delivery systems are those which can providefor release of the active component of the invention in sustainedrelease pellets or capsules. Many types of sustained release deliverysystems are available. These include, but are not limited to: (a)erosional systems in which the active component is contained within amatrix, and (b) diffusional systems in which the active componentpermeates at a controlled rate through a polymer.

The present invention also provides the use of high density lipoprotein(HDL) in the manufacture of a medicament for parenteral administrationto a patient for the treatment and/or prophylaxis of an inflammatorycondition of the intestine of the patient.

In yet another aspect, the invention provides an agent for parenteraladministration in the treatment and/or prophylaxis of an inflammatorycondition of the intestine of a patient, which comprises high densitylipoprotein (HDL).

The present invention is further illustrated by the followingnon-limiting Examples.

EXAMPLES

Materials and Methods

Animals

Male Sprague-Dawley rats (300-350 g; Charles River; Milan; Italy) werehoused in a controlled environment and provided with standard rodentchow and water. Animal care was in compliance with Italian regulationson protection of animals used for experimental and other scientificpurpose (D.M. 116192) as well as with the EEC regulations (O.J. of E.C.L 358/1 Dec. 18, 1986).

Experimental Groups (Colitis Study)

Upon completion of surgical procedures, rats were randomly allocatedinto the following four groups: (i) DNBS+saline group; rats were givenDNBS (100 mg/kg, i.c.) (N=10), (ii) DNBS+recHDL group; rats weresubjected to identical procedures as above and administered recHDL (40mg/kg/day, i.v.) for 3 days (N=10), (iii) Sham+saline group;(sham-operated) rats were subjected to identical procedures as aboveexcept that the vehicle alone (50% ethanol, 0.8 ml) was injected insteadof DNBS and were maintained under anaesthesia for the duration of theexperiment (N=10), (vi) Sham+HDL group; identical to sham-operated ratsexcept for the administration of recHDL (40 mg/kg/day, i.v.) (N=10). Thedose of recHDL used in the present study was taken from previous studiesshowing efficacy in models of renal I/R injury (11).

Experimental Groups (SAO Study)

Upon completion of surgical procedures, rats were randomly allocatedinto the following four groups: (i) I/R+saline group; rats weresubjected to SAO shock (45 min) followed by reperfusion (6 h) (N=10),(ii) I/R+rec HDL group; rats were subjected to identical surgicalprocedures as above and administered recHDL (80 mg/kg, i.v.) 30 minprior to commencement of I/R (N=10), (iii) Sham+saline group;(sham-operated) rats were subjected to identical surgical proceduresexcept for SAO shock and were maintained under anaesthesia for theduration of the experiment (N=10), (vi) Sham+recHDL group; identical tosham-operated rats except for the administration of recHDL (80 mg/kg,i.v.) 30 min prior to commencing I/R (N=10).

Induction of Experimental Colitis

Colitis was induced by using a technique of acid-induced coloninflammation as described previously (23). In fasted rats lightlyanaesthetised with isoflurane, a 3.5 F catheter was inserted into thecolon via the anus until approximately the splenic flexure (8 cm fromthe anus). DNBS (100 mg/kg i.c.) was dissolved in 50% ethanol (totalvolume, 0.8 ml). Thereafter, the animals were kept for 15 minutes in aTrendelenburg position to avoid reflux. After colitis and sham-colitisinduction, the animals were observed for 3 days. On Day 4, the animalswere weighed and anaesthetised with chloral hydrate (400 mg/kg, i.p.),and the abdomen was opened by a midline incision. The colon was removed,freed from surrounding tissues, opened along the antimesenteric border,rinsed, weighed, and processed for histology and immunohistochemistry.The macroscopic damage score, according to Wallace et al. (23) wasassessed.

Splanchnic Artery Occlusion Shock (SAO-Shock)

Male Sprague-Dawley rats were anaesthetised with sodium pentobarbital(45 mg/kg, i.p.). Following anaesthesia, catheters were placed in thecarotid artery and jugular vein as described previously (13). Bloodpressure was monitored continuously by a Maclab A/D converter (ADInstruments), and stored and displayed on a Macintosh personal computer.After midline laparotomy, the celiac and superior mesenteric arterieswere isolated near their aortic origins. During this procedure, theintestinal tract was maintained at 37° C. by placing it between gauzepads soaked with warmed 0.9% NaCl solution.

Rats were observed for a 30 min stabilisation period before eithersplanchnic ischaemia or sham ischaemia. SAO shock was induced byclamping both the superior mesenteric artery and the celiac trunk,resulting in a total occlusion of these arteries for 45 min. After thisperiod of occlusion, the clamps were removed. In one study, the variousgroups of rats were sacrificed 60 min after the commencement ofreperfusion for histological examination of the bowel and forbiochemical studies, as described below. In another set of studies, thevarious groups of rats were observed for 6 h following reperfusion inorder to determine survival differences.

Reconstituted High Density Lipoprotein (recHDL).

The discoidal recHDLs were provided by ZLB-Bioplasma, Bern, Switzerland.The particles, containing human apo A-I as the sole protein and soybeanphosphatidylcholine as the sole phospholipid were prepared using cholatedialysis (24). Their physicochemical properties have been described indetail (25).

Light Microscopy

Ileum was collected after 1 h of reperfusion from the rats subjected toSAO shock. The colon was collected 4 days after DNBS administration.After fixation for 1 week at room temperature in Dietrich solution(14.25% ethanol, 1.85% formaldehyde, 1% acetic acid), samples weredehydrated in graded ethanol and embedded in Paraplast (SherwoodMedical, Mahwah, N.J.). Thereafter, 7-mm sections were deparaffinizedwith xylene, stained with haematoxylin-eosin and trichromic vanGiesson's stain, and observed in a Dialux 22 Leitz (Wetziar, Germany)microscope. Colon damage was scored by two independent observers asdescribed previously (26), according to the following morphologicalcriteria: 0, no damage; 1, localised hyperaemia without ulcers; 2,linear ulcers with no significant inflammation; 3, linear ulcers withinflammation at one site; 4, two or more major sites of inflammation andulceration extending>1 cm along the length of the colon; and 5-8, onepoint is added for each centimetre of ulceration beyond an initial 2 cm.

Immunohistochemical Localisation for ICAM-I, Nitrotyrosine and Poly(ADP-Ribose) (PAR).

At the specified time, the ileum and the colon tissues were fixed in 10%(w/v) phosphate buffered saline (PBS)-buffered formaldehyde and 8 μmsections were prepared from paraffin embedded tissues. Afterdeparaffinization, endogenous peroxidase was quenched with 0.3% (v/v)hydrogen peroxide in 60% (v/v) methanol for 30 min. The sections werepermeablised with 0.1% (w/v) Triton X-100 in PBS for 20 min.Non-specific adsorption was minimised by incubating the sections in 2%(v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidinbinding sites were blocked by sequential incubation for 15 min withbiotin and avidin (DBA, Milan, Italy), respectively. Sections wereincubated overnight with anti-nitrotyrosine rabbit polyclonal antibody(1:500 in PBS, v/v) or with anti-poly (ADP-ribose) goat polyclonalantibody (1:500 in PBS, v/v) or with mouse anti-rat antibody directed atICAM-1 (CD54) (1:500 in PBS, v/v) (DBA, Milan, Italy). Sections werewashed with PBS, and incubated with secondary antibody. Specificlabelling was detected with a biotin-conjugated goat anti-rabbit IgG andavidin-biotin peroxidase complex (DBA, Milan, Italy). To verify thebinding specificity for ICAM-1 and PAR, some sections were alsoincubated with primary antibody only (no secondary antibody) or withsecondary antibody only (no primary antibody). In these situations, nopositive staining was found in the sections indicating that theimmunoreactions were positive in all the experiments carried out. Inorder to confirm that the immunoreactions for the nitrotyrosine werespecific some sections were also incubated with the primary antibody(anti-nitrotyrosine) in the presence of excess nitrotyrosine (10 mM) toverify the binding specificity. Immunocytochemistry photographs (N=5)were assessed by densitometry as previously described (27) by usingOptilab Graftek software on a Macintosh personal computer.

Myeloperoxidase (MPO) Activity

MPO activity, an indicator of PMN accumulation, was determined aspreviously described (28). At the specified time point the ileum and thecolon were removed and weighed. The tissues were homogenised in asolution containing 0.5% hexa-decyl-trimethyl-ammonium bromide and 10 mM3-(N-morpholino)-propane-sulfonic acid dissolved in 80 mM sodiumphosphate buffer (pH 7), and centrifuged for 30 min at 20,000 g at 4° C.An aliquot of the supernatant was then allowed to react with a solutionof tetra-methyl-benzidine (16 mM) and 1 mM hydrogen peroxide. The rateof change in absorbance was measured by a spectrophotometer at 650 nm.MPO activity was defined as the quantity of enzyme degrading 1 μmol ofperoxide/min at 37° C. and was expressed in units per gram weight of wettissue.

Malondialdehyde (MDA) Measurement

The levels of MDA in the ileum and colon were determined as an indicatorof lipid peroxidation (29). At the specified time point the ileum andthe colon were removed, weighed and homogenised in 1.15% KCl solution.An aliquot (100 μl) of the homogenate was added to a reaction mixturecontaining 200 μl of 8.1% SDS, 1500 μl of 20% acetic acid (pH 3.5), 1500μl of 0.8% thiobarbituric acid and 700 μl distilled water. Samples werethen boiled for 1 h at 95° C. and centrifuged at 3,000×g for 10 min. Theabsorbance of the supernatant was measured by spectrophotometry at 650nm.

Measurement of Cytokines

The levels of TNF-α and IL-1β were evaluated in the plasma collected 60min after reperfusion in the SAO model and in the colon tissues at 4days after intra-colonic injection of DNBS. The assay was carried out byusing a colorimetric, commercial kit (Calbiochem-NovabiochemCorporation, USA). The ELISA (Enzyme-Linked Immunosorbent Assay) has alower detection limit of 5 pg/ml.

Materials.

Biotin blocking kit, biotin-conjugated goat anti-rabbit IgG andavidin-biotin peroxidase complex were obtained from Vector Laboratories(Burlingame, Calif., USA). Primary anti-nitrotyrosine antibody waspurchased from Upstate Biotech (Saranac Lake, N.Y.). Primary ICAM-1(CD54) was purchased from Pharmingen (DBA, Milan, Italy). All otherreagents and compounds used were purchased from Sigma Chemical Company(Sigma, St. Louis, Mo.).

Data Analysis

All values in the figures and text are expressed as mean±standard error(s.e.m.) of the mean of N observations. For the in vivo studies Nrepresents the number of animals studied. In the experiments involvinghistology or immunohistochemistry, the figures shown are representativeof at least three experiments performed on different experimental days.The results were analysed by one-way ANOVA followed by a Bonferronipost-hoc test for multiple comparisons. A p-value less of than 0.05 wasconsidered significant.

Results

Protective Effects of HDL in Splanchnic Artery Occlusion Shock

Occlusion of the splanchnic arteries produced an increase in MAP, whichthen decreased until death occurred (FIG. 1A). The mean survival timewas found to be 74±1.7 min, whereas control sham animals survived forthe entire period of the experiment (FIG. 1B). Administration of recHDLsignificantly prevented the fall in blood pressure seen afterreperfusion and increased the survival rate (FIG. 1). Having establishedthe survival time, in another series of experiments, animals weresacrificed either after the period of ischaemia or 60 minutespost-reperfusion in order to collect blood and tissues for biochemicalanalysis. Reperfusion of the ischaemic splanchnic circulation led to thefollowing events: a substantial increase in intestinal lipidperoxidation products as determined by increased levels of MDA (FIG.2A), TNF-α and IL-1β (FIG. 3), and a profound infiltration of PMNs intothe intestine as determined by MPO activity (FIG. 2B). Ileum sectionscollected from SAO-shocked rats at 60 min of reperfusion showed anincrease of positive staining for ICAM-1 along vessels and in thenecrotic tissue (FIGS. 4A, 5A). These inflammatory events were triggeredby the reperfusion phase since no changes were observed when blood ortissues were removed after the period of ischaemia alone (FIG. 5A).recHDL (80 mg/kg), when given i.v. 30 min prior to ischaemia,significantly inhibited the increased levels of MDA in the ileum (FIG.2A) as well as TNF-α and IL-1β (FIG. 3). HDL significantly reduced thePMN infiltration into the ileum (FIG. 2B) and the up-regulation ofICAM-1, which was expressed in the endothelium along the vascular wall(FIGS. 4B, 5A). Staining of ileum sections obtained from sham-operatedrats with anti-ICAM-1 antibody showed a specific staining along vessels,demonstrating that ICAM-1 is constitutively expressed (FIG. 5A). Inaddition, ileum sections obtained from SAO-shocked rats at 60 min ofreperfusion showed positive staining for nitrotyrosine (FIGS. 5A, 6A)and PAR (FIGS. 5A, 6C). Administration of recHDL reduced the degree ofimmunostaining for nitrotyrosine (FIG. 5A, 6B) and PAR (FIG. 5A, 6D) inthe repressed intestine. No staining for either nitrotyrosine or PAR wasobserved in sham-operated rats (FIG. 5A). Finally, histologicalexaminations of the small intestine at 60 min post reperfusion (seerepresentative sections in FIG. 7) revealed the following pathologicalchanges: Ileum sections showed inflammatory infiltration by inflammatorycells extending through the wall and concentrated below the epitheliallayer (FIG. 7A). recHDL treated rats show a significant reduction inorgan injury (FIG. 7B). No histological alterations were observed insham-treated rats (data not shown).

Protective Effects of HDL in DNBS-Induced Colitis

Four days after intra-colonic administration of DNBS, the colon appearedflaccid and filled with liquid stool. The macroscopic inspection ofcecum, colon and rectum showed presence of mucosal congestion, erosionand haemorrhagic ulcerations (FIG. 8A). The histopathological featuresincluded a transmural necrosis and oedema and a diffuse PMN cellularinfiltrate in the submucosa (FIG. 8B). The inflammatory changes of theintestinal tract were associated with an increase in the weight of thecolon (FIG. 9A). Treatment of rats with recHDL (40 mg/kg/day)significantly attenuated the extent and severity of the histologicalsigns of colon injury (FIGS. 8A, 8C). A significant increase in theweight of the spleen, an indicator of inflammation, was also noted invehicle-treated rats, which had received DNBS (FIG. 9B). No significantincrease in weight of either colon or spleen was observed in DNBS-rats,which had been treated with recHDL (FIG. 9). The severe colitis causedby DNBS was associated with a significant loss in body weight (FIG.10A). Treatment of DNBS-rats with recHDL significantly reduced the lossin body weight (FIG. 10A). Colonic injury by DNBS administration wasalso characterised by an increase of pro-inflammatory cytokines (TNF-α,and IL-1β) in the colon (FIG. 10B). recHDL treatment reduced theincrease in TNF-α and IL-1β as observed in colonic tissues (FIG. 10B).

At 4 days after DNBS treatment, sections of colon from sham-administeredrats did not stain for either nitrotyrosine or PAR (FIG. 5B). Colonsections obtained from vehicle-treated DNBS rats exhibited positivestaining for nitrotyrosine (FIG. 11A) and PAR (FIG. 11C), which waslocalised in inflammatory cells and in disrupted epithelial cells.recHDL treatment reduced the degree of immunostaining for nitrotyrosine(FIG. 11 B) and PAR (FIG. 11D) in the colon of DNBS-treated rats. Thepresence of nitrotyrosine staining in the colon correlated positivelywith the increase in tissue levels of MDA, indicating an increase inlipid peroxidation (FIG. 12A). recHDL treatment significantly reducedthe degree of lipid peroxidation (determined as a decrease in tissue MDAlevels, FIG. 12A). The colitis caused by DNBS was also characterised byan increase in MPO activity (FIG. 12B). This finding is consistent withthe observation made with light microscopy that the colon ofvehicle-treated DNBS rats contained a large number of PMNs. Infiltrationof PMNs into the mucosa has been suggested to contribute significantlyto the tissue necrosis and mucosal dysfunction associated with colitis,as activated PMNs release large amounts of free radicals. To furtherelucidate the effect of recHDL treatment on PMN accumulation in theinflamed colon, we next evaluated the intestinal expression of ICAM-1.Tissue sections obtained from sham-operated rats with anti-ICAM-1antibody showed a specific staining along the vessels, demonstratingthat ICAM-1 is expressed constitutively in endothelial cells (FIG. 5B).After DNBS administration, the staining intensity substantiallyincreased in the vessels of the lamina propria and submucosa.Immunohistochemical staining for ICAM-1 was also present in epithelialcells of injured colon and in infiltrated inflammatory cells in damagedtissues from DNBS-treated rats (FIG. 13B). Treatment of DNBS-rats withrecHDL, however, significantly reduced both the degree of PMNinfiltration (determined as a decrease in MPO activity, FIG. 12B) andthe up-regulation of the constitutive ICAM-1, which was normallyexpressed in the endothelium along the vascular wall (FIG. 13B).

Discussion

This study provides evidence that treatment of rats with recHDLattenuates: (i) the development of SAO shock, (ii) the infiltration ofthe ileum with PMNs (histology and MPO activity), (iii) the degree oflipid peroxidation in the ileum, and (iv) the degree of ileum injury(histology) caused by ischaemia and reperfusion; (v) the development ofDNBS-induced colitis, (vi) the infiltration of the colon with PMNs(histology and MPO activity), (vii) the degree of lipid peroxidation inthe colon, and (viii) the degree of colon injury (histology) in ratstreated with DNBS. All of these findings support the view that recHDLattenuates the degree of gut inflammation in the rat.

HDL Reduces Oxidative and Nitrosative Damage Associated with GutIschaemia and with DNBS-Induced Colitis

Reactive oxygen and nitrogen species play a key role in gut injuryassociated with I/R (12,13,30) and with IBD (14,31). The importantcontribution to the pathophysiology of IBD of reactive oxygen ornitrogen species has been demonstrated in both animal and clinicalstudies. These species are cytotoxic agents, inducing lipid peroxidationand other cellular oxidative stress by cross linking proteins, lipids,and nucleic acids, which then cause cellular dysfunction, damage, andeventually death. Evidence consistent with damage caused by reactiveradical species is provided by a large number of animal and clinicalstudies (12,13,30,31). In the present study, it has been found that theintestinal damage induced either by I/R or by intra-colonicadministration of DNBS was associated with high concentrations of MDA,which is considered a good indicator of lipid peroxidation (29). Recentevidence indicates that nitration of tyrosine can result from a numberof chemical actions, and can be considered as a global marker ofnitrosative stress (32). Nitrotyrosine can be formed from the reactionof nitrite with hypochlorous acid or the reaction of nitrite with MPOand hydrogen peroxide (33). In these experiments, increasedimmunohistochemical expression of nitrotyrosine has been found mostlylocalised on epithelial cells and in the area of infiltratedinflammatory cells, suggesting that peroxynitrite or other nitrogenderivatives and oxidants are formed in vivo and may contribute to tissueinjury. These data are consistent with previous findings thatimmunohistochemical staining for nitrotyrosine was localised oninflammatory cells during DNBS-induced colitis (34) and during SAO-shock(13,27). The pathogenic role of nitrogen derived species such asperoxynitrite (35) in gut inflammation is further supported by the factthat intra-colonic administration of exogenous peroxynitrite induces asevere colonic inflammation, which mimics the features of bothulcerative colitis and Crohn's disease. In the present study it wasobserved that ileum and colon injury was significantly less in ratstreated with recHDL. Indeed, HDL treatment prevented the formation oftissue MDA and nitrotyrosine staining in SAO-shocked rats andDNBS-treated animals. ROS [reactive oxygen species] cause single-stranddamage, leading to PARP activation and cell death (36). Some evidenceexists to support the possible role of PARP activation in thepathophysiology of gut inflammation (37). recHDL treatment reduced PARformation, an index of PARP activation, and this effect may wellcontribute to the overall protective effects of HDLs observed in thesestudies.

The Beneficial Effect of HDLs in SAO-Shock and in DNBS-Induced Colitisis Related to an Inhibition of Cytokines Production

TNF-α and IL-1β are clearly involved in the pathogenesis of I/R andcolitis and these cytokines are present in the colon during inflammation(13,27,38). Direct evidence that TNF-α and IL-1β play a role in thepathogenesis of experimental colitis and SAO-shock has been obtained inanimal models in which blocking of the action of these cytokines hasbeen shown to delay the onset of gut injury and suppress the associatedinflammatory response (13,27). A role for TNF-α in human disease camefrom recent studies using Infliximab (38), a chimeric anti-TNF antibody,and CDP571, a humanised monoclonal antibody to TNF-α, and membrane-boundTNF without fixing complement nor mediating antibody-dependent cellularcytotoxicity (40). In both cases, significant reduction in Crohn'sdisease activity index (CDAI) as well as attenuation ofhistopathological and endoscopic inflammation in Crohn's diseasepatients was observed.

As the present study demonstrates that recHDL significantly reduces therelease of TNF-α and IL-1β associated with SAO-shock and withDNBS-induced colitis, and without wishing to be bound by any one theory,it is postulated by the present inventors that the inhibition of theformation of TNF-α and IL-1β plays an important role in the overallbeneficial effects observed with HDL. These results are in agreementwith previous observations, which show that HDL also attenuates therelease of pro-inflammatory cytokines (including TNF-α) caused by smalldoses of intravenously administered LPS (10).

The Beneficial Effects of HDL in SAO-Shock and in DNBS-Induced Colitisare Related to an Alteration in PMN Recruitment.

PMNs play a crucial role in the development and full manifestation ofgastrointestinal inflammation, as they represent a major source of freeradicals in the inflamed intestinal mucosa (41). PMN infiltration intoinflamed tissues plays a crucial role in the destruction of foreignantigens and in the breakdown and remodelling of injured tissue (42).The interactions of PMNs with the endothelium are regulated by variousadhesion molecules including the selectins, the Beta2 integrins andadhesion molecules of the immunoglobulin superfamily (43). A firmadherence of the PMN to the endothelial surface is required fortransendothelial migration (21). This firm adherence involves theinteraction of Beta2 integrins (i.e., CD11/CD18) on the PMN surface andICAM-1 on the endothelial cell surface (44).

A major finding of this study was that, not only did the recHDL-treatedrats show a remarkable recovery of the intestinal injury associated witha reduction in oxidative and nitrosative damage after I/R as well asafter DNBS administration, but also in recHDL-treated rats, infiltrationof PMNs into the colon was significantly reduced. Furthermore, ICAM-1was expressed in endothelial and epithelial cells, and PMNs both in theileum from SAO-shocked rats and in the distal colon from DNBS-treatedrats. recHDL treatment was associated with a significant reduction ofICAM-1 expression in endothelial and epithelial cells. These resultsclearly demonstrate that recHDLs can interrupt the cascade of eventsleading to the interaction between PMNs and endothelial cells in thelate firm adhesion phase mediated by ICAM-1, and these findings are inagreement with other previously published studies (9,10).

Conclusions

These results clearly demonstrate that recHDLs are protective inSAO-shock and in experimental colitis, and that inhibition of TNF-α andIL-1β formation (amongst other effects which include inhibition of PMNinfiltration) in the injured tissue probably accounts for the beneficialeffects.

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1. A method for the treatment and/or prophylaxis of an inflammatorycondition of the intestine of a patient, which comprises parenteraladministration to the patient of an effective amount of high densitylipoprotein (HDL).
 2. The method of claim 1, wherein said patient is ahuman.
 3. The method of claim 1, wherein said inflammatory condition isischaemia/reperfusion (I/R) injury of the intestine, inflammatory boweldisease, acute infective colitis, or pseudomembranous colitis.
 4. Themethod of claim 1, wherein said HDL is selected from the groupconsisting of nascent HDL, reconstituted HDL, recombinant HDL and afunctional peptide or other analogue thereof.
 5. The method of claim 4,wherein said HDL is reconstituted HDL.
 6. The method of claim 1, whereinsaid HDL is administered in a dosage range of from 0.1-200 mg per kgbody weight of the patient per treatment, preferably in a dosage rangeof from 10-80 mg per kg body weight per treatment.
 7. The method ofclaim 1, wherein said parenteral administration is selected from thegroup consisting of intravenous, intraarterial, intramuscular andsubcutaneous injection or infusion.
 8. The method of claim 7, whereinsaid parenteral administration is intravenous injection or infusion. 9.The use of high density lipoprotein (HDL) in the manufacture of amedicament for parenteral administration to a patient for the treatmentand/or prophylaxis of an inflammatory condition of the intestine of thepatient.
 10. The use of claim 9, wherein said patient is a human. 11.The use of claim 9, wherein said inflammatory condition isischaemia/reperfusion (I/R) injury of the intestine, inflammatory boweldisease, acute infective colitis, or pseudomembranous colitis.
 12. Theuse of claim 9, wherein said HDL is selected from the group consistingof nascent HDL, reconstituted HDL, recombinant HDL and a functionalpeptide or other analogue thereof.
 13. The use of claim 12, wherein saidHDL is reconstituted HDL.
 14. The use of claim 9, wherein said HDL isadministered in a dosage range of from 0.1-200 mg per kg body weight ofthe patient per treatment, preferably in a dosage range of from 10-80 mgper kg body weight per treatment.
 15. The use of claim 9, wherein saidparenteral administration is selected from the group consisting ofintravenous, intraarterial, intramuscular and subcutaneous injection orinfusion.
 16. The use of claim 15, wherein said parenteraladministration is intravenous injection or infusion.
 17. An agent forparenteral administration in the treatment and/or prophylaxis of aninflammatory condition of the intestine of a patient, which compriseshigh density lipoprotein (HDL).